Blocking Tim-3 or/and PD-1 reverses dysfunction of tumor-infiltrating lymphocytes in HBV-related hepatocellular carcinoma.

The immunosuppression of tumor-infiltrating lymphocytes (TILs) is associated with rapid progression of hepatitis B virus-related hepatocellular carcinoma (HBV-HCC). T cell Ig- and mucin-domain-containing molecule-3 (Tim-3) and programmed cell death 1 (PD-1) are important inhibitory molecules expressed on the surface of T cells, but their roles in the function of TILs in HBV-HCC are poorly understood. We aimed to study the roles of these two markers in HBV-HCC. Ninety patients with pathologically confirmed HBV-associated HCC were enrolled in our study. Blood samples, paired fresh tumor tissues and adjacent tissues were collected, and isolating peripheral blood mononuclear cells, TILs and adjacent-infiltrating lymphocytes were isolated from these samples. The patients were followed-up to allow survival analysis. Tim-3 or/and PD-1 was up-regulated expressed on CD4+ and CD8+ TILs in HBV-HCC patients and a higher proportion of TILs expressed PD-1 alone. Tim-3+ and PD-1+ TILs greatly decreased secretion of IFN-? and TNF-a. Expression of Tim-3 and PD-1 on TILs negatively correlated with disease-free survival of HCC patients. Direct blockade of Tim-3 and PD-1 in vitro significantly enhanced TILs proliferation and secretion of IFN-? and TNF-a. Expression of Tim-3 and/or PD-1 on TILs impairs their function and correlates negatively with disease-free survival in HBV-HCC. Direct blockade of Tim-3 and PD-1 restores anti-tumor effects of TILs, which suggests a potential target for novel immunotherapy in HBV-HCC.

Liu F ，Zeng G ，Zhou S ，He X ，Sun N ，Zhu X ，Hu A ... -  《-》

Tim-3/galectin-9 signaling pathway mediates T-cell dysfunction and predicts poor prognosis in patients with hepatitis B virus-associated hepatocellular carcinoma.

The interaction between T cell immunoglobulin- and mucin-domain-containing molecule (Tim-3) expressed on T helper 1 (Th1) cells, and its ligand, galectin-9, negatively regulates Th1-mediated immune responses. However, it is poorly understood if and how the Tim-3/galectin-9 signaling pathway is involved in immune escape in patients with hepatocellular carcinoma (HCC). Here we studied the expression, function, and regulation of the Tim-3/galectin-9 pathway in patients with hepatitis B virus (HBV)-associated HCC. We detected different levels of galectin-9 expression on antigen-presenting cell (APC) subsets including Kupffer cells (KCs), myeloid dendritic cells (DCs), and plasmacytoid DCs in HCC. The highest galectin-9 expression was on KCs in HCC islets, not in the adjacent tissues. Furthermore, Tim-3 expression was increased on CD4(+) and CD8(+) T cells in HCC as compared to the adjacent tissues, and Tim-3(+) T cells were replicative senescent and expressed surface and genetic markers for senescence. Interestingly, tumor-infiltrating T-cell-derived interferon (IFN)-γ stimulated the expression of galectin-9 on APCs in the HCC microenvironment. Immunofluorescence staining revealed a colocalization of Tim-3(+) T cells and galectin-9(+) KCs in HCC. Functional studies demonstrated that blockade of the Tim-3/galectin-9 signaling pathway importantly increased the functionality of tumor-infiltrating Tim-3(+) T cells as shown by increased T-cell proliferation and effector cytokine production. Finally, we show that the numbers of Tim-3(+) tumor-infiltrating cells were negatively associated with patient survival. Our work demonstrates that the Tim-3/galectin-9 signaling pathway mediates T-cell senescence in HBV-associated HCC. The data suggest that this pathway could be an immunotherapeutic target in patients with HBV-associated HCC.

Li H ，Wu K ，Tao K ，Chen L ，Zheng Q ，Lu X ，Liu J ，Shi L ，Liu C ，Wang G ，Zou W ... -  《-》

Antibodies Against Immune Checkpoint Molecules Restore Functions of Tumor-Infiltrating T Cells in Hepatocellular Carcinomas.

Ligand binding to inhibitory receptors on immune cells, such as programmed cell death 1 (PD-1) and cytotoxic T-lymphocyte associated protein 4 (CTLA4), down-regulates the T-cell-mediated immune response (called immune checkpoints). Antibodies that block these receptors increase antitumor immunity in patients with melanoma, non-small-cell lung cancer, and renal cell cancer. Tumor-infiltrating CD4+ and CD8+ T cells in patients with hepatocellular carcinoma (HCC) have been found to be functionally compromised. We analyzed HCC samples from patients to determine if these inhibitory pathways prevent T-cell responses in HCCs and to find ways to restore their antitumor functions. We collected HCC samples from 59 patients who underwent surgical resection from November 2013 through May 2017, along with tumor-free liver tissues (control tissues) and peripheral blood samples. We isolated tumor-infiltrating lymphocytes (TIL) and intra-hepatic lymphocytes. We used flow cytometry to quantify expression of the inhibitory receptors PD-1, hepatitis A virus cellular receptor 2 (TIM3), lymphocyte activating 3 (LAG3), and CTLA4 on CD8+ and CD4+ T cells from tumor, control tissue, and blood; we studied the effects of antibodies that block these pathways in T-cell activation assays. Expression of PD-1, TIM3, LAG3, and CTLA4 was significantly higher on CD8+ and CD4+ T cells isolated from HCC tissue than control tissue or blood. Dendritic cells, monocytes, and B cells in HCC tumors expressed ligands for these receptors. Expression of PD-1, TIM3, and LAG3 was higher on tumor-associated antigen (TAA)-specific CD8+ TIL, compared with other CD8+ TIL. Compared with TIL that did not express these inhibitory receptors, CD8+ and CD4+ TIL that did express these receptors had higher levels of markers of activation, but similar or decreased levels of granzyme B and effector cytokines. Antibodies against CD274 (PD-ligand1 [PD-L1]), TIM3, or LAG3 increased proliferation of CD8+ and CD4+ TIL and cytokine production in response to stimulation with polyclonal antigens or TAA. Importantly, combining antibody against PD-L1 with antibodies against TIM3, LAG3, or CTLA4 further increased TIL functions. The immune checkpoint inhibitory molecules PD-1, TIM3, and LAG3 are up-regulated on TAA-specific T cells isolated from human HCC tissues, compared with T cells from tumor-free liver tissues or blood. Antibodies against PD-L1, TIM3, or LAG3 restore responses of HCC-derived T cells to tumor antigens, and combinations of the antibodies have additive effects. Strategies to block PD-L1, TIM3, and LAG3 might be developed for treatment of primary liver cancer.

Zhou G ，Sprengers D ，Boor PPC ，Doukas M ，Schutz H ，Mancham S ，Pedroza-Gonzalez A ，Polak WG ，de Jonge J ，Gaspersz M ，Dong H ，Thielemans K ，Pan Q ，IJzermans JNM ，Bruno MJ ，Kwekkeboom J ... -  《-》

[Up-regulation of TIM-3 on CD4+ tumor infiltrating lymphocytes predicts poor prognosis in human non-small-cell lung cancer].

Ji P ，Chen D ，Bian J ，Xia R ，Song X ，Wen W ，Zhang X ，Zhu Y ... -  《-》

TIGIT and PD1 Co-blockade Restores ex vivo Functions of Human Tumor-Infiltrating CD8(+) T Cells in Hepatocellular Carcinoma.

TIGIT is a co-inhibitory receptor, and its suitability as a target for cancer immunotherapy in HCC is unknown. PD1 blockade is clinically effective in about 20% of advanced HCC patients. Here we aim to determine whether co-blockade of TIGIT/PD1 has added value to restore functionality of HCC tumor-infiltrating T cells (TILs). Mononuclear leukocytes were isolated from tumors, paired tumor-free liver tissues (TFL) and peripheral blood of HCC patients, and used for flow cytometric phenotyping and functional assays. CD3/CD28 T-cell stimulation and antigen-specific assays were used to study the ex vivo effects of TIGIT/PD1 single or dual blockade on T-cell functions. TIGIT was enriched, whereas its co-stimulatory counterpart CD226 was down-regulated on PD1high CD8+ TILs. PD1high TIGIT+ CD8+ TILs co-expressed exhaustion markers TIM3 and LAG3 and demonstrated higher TOX expression. Furthermore, this subset showed decreased capacity to produce IFN-γ and TNF-α. Expression of TIGIT-ligand CD155 was up-regulated on tumor cells compared with hepatocytes in TFL. Whereas single PD1 blockade preferentially enhanced ex vivo functions of CD8+ TILs from tumors with PD1high CD8+ TILs (high PD1 expressers), co-blockade of TIGIT and PD1 improved proliferation and cytokine production of CD8+ TILs from tumors enriched for PD1int CD8+ TILs (low PD1 expressers). Importantly, ex vivo co-blockade of TIGIT/PD1 improved proliferation, cytokine production, and cytotoxicity of CD8+ TILs compared with single PD1 blockade. Ex vivo, co-blockade of TIGIT/PD1 improves functionality of CD8+ TILs that do not respond to single PD1 blockade. Therefore co-blockade of TIGIT/PD1 could be a promising immune therapeutic strategy for HCC patients.

Ge Z ，Zhou G ，Campos Carrascosa L ，Gausvik E ，Boor PPC ，Noordam L ，Doukas M ，Polak WG ，Terkivatan T ，Pan Q ，Takkenberg RB ，Verheij J ，Erdmann JI ，IJzermans JNM ，Peppelenbosch MP ，Kraan J ，Kwekkeboom J ，Sprengers D ... -  《Cellular and Molecular Gastroenterology and Hepatology》